Transgenic plants with enhanced agronomic traits

ABSTRACT

This disclosure describes screening a population of transgenic plants derived from plant cells transformed with recombinant DNA for expression of proteins with homeobox domains to identify plant cells of specific transgenic events that are useful for imparting enhanced traits to transgenic crop plants. Traits include enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, enhanced tolerance to cold stress and/or improved seed compositions. Also disclosed are transgenic seeds for growing a transgenic plant having the recombinant DNA in its genome and exhibiting the screened enhance trait. Also disclosed are methods for generating seed and plants based on the transgenic events.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 USC §119(e) of U.S. provisionalapplication Ser. No. 60/638,099, filed Dec. 21, 2004, incorporatedherein by reference.

INCORPORATION OF SEQUENCE LISTING

A sequence listing and a computer readable form (CRF) of the sequencelisting, on CD-ROM, each containing the text file named“G1543C.ST25.txt”, which is 63 KB (measured in MS-WINDOWS) and wascreated on Dec. 18, 2005, are herein incorporated by reference.

INCORPORATION OF COMPUTER LISTING

Appended hereto is a Computer Listing on duplicate CD-ROMs containing afolder labeled “hmmer-2.3.2” and two _.HMM files, incorporated herein byreference. Folder hmmer-2.3.2 contains the source code and otherassociated files for implementing the HMMer software for Pfam analysis.The _.HMM files contains Pfam Hidden Markov Models. The ComputerListings were created on Dec. 18, 2005.

FIELD OF THE INVENTION

Disclosed herein are inventions in the field of plant genetics anddevelopmental biology. More specifically, the inventions provide plantcells with recombinant DNA for providing an enhanced trait in atransgenic plant, plants comprising such cells, seed and pollen derivedfrom such plants, methods of making and using such cells, plants, seedsand pollen. In particular, the recombinant DNA of the inventions expresstranscription factors with homeobox domains.

BACKGROUND OF THE INVENTION

Transgenic plants with improved agronomic traits such as yield,environmental stress tolerance, pest resistance, herbicide tolerance,improved seed compositions, and the like are desired by both farmers andconsumers. Although considerable efforts in plant breeding have providedsignificant gains in desired traits, the ability to introduce specificDNA into plant genomes provides further opportunities for generation ofplants with improved and/or unique traits. Merely introducingrecombinant DNA into a plant genome doesn't always produce a transgenicplant with an enhanced agronomic trait. Methods to select individualtransgenic events from a population are required to identify thosetransgenic events that are characterized by the enhanced agronomictrait.

SUMMARY OF THE INVENTION

This invention employs recombinant DNA for expression of proteins thatare useful for imparting enhanced agronomic traits to the transgenicplants. Recombinant DNA in this invention is provided in a constructcomprising a promoter that is functional in plant cells and that isoperably linked to DNA that encodes a protein having domains of aminoacids in a sequence that exceed the Pfam gathering cutoff for amino acidsequence alignment with a Pfam Homeobox protein domain family and a PfamHALZ protein domain family. The Pfam gathering cuttoff for the Homeoboxprotein domain family is −4 and the Pfam gathering cuttoff for the HALZprotein domain family is 17. Other aspects of the invention arespecifically directed to transgenic plant cells comprising therecombinant DNA of the invention, transgenic plants comprising aplurality of such plant cells, progeny transgenic seed and transgenicpollen from such plants. Such plant cells are selected from a populationof transgenic plants regenerated from plant cells transformed withrecombinant DNA and that express the protein by screening transgenicplants in the population for an enhanced trait as compared to controlplants that do not have said recombinant DNA, where the enhanced traitis selected from group of enhanced traits consisting of enhanced wateruse efficiency, enhanced cold tolerance, increased yield, enhancednitrogen use efficiency, enhanced seed protein and enhanced seed oil.

In yet another aspect of the invention the plant cells, plants, seedsand pollen further comprise DNA expressing a protein that providestolerance from exposure to an herbicide applied at levels that arelethal to a wild type of said plant cell. Such tolerance is especiallyuseful not only as an advantageous trait in such plants but is alsouseful in a selection step in the methods of the invention. In aspectsof the invention the agent of such herbicide is a glyphosate, dicamba,or glufosinate compound.

Yet other aspects of the invention provide transgenic plants which arehomozygous for the recombinant DNA and transgenic seed of the inventionfrom corn, soybean, cotton, canola, alfalfa, wheat or rice plants. Inother important embodiments for practice of various aspects of theinvention in Argentina the recombinant DNA is provided in plant cellsderived from corn lines that that are and maintain resistance to the Malde Rio Cuarto virus or the Puccina sorghi fungus or both.

This invention also provides methods for manufacturing non-natural,transgenic seed that can be used to produce a crop of transgenic plantswith an enhanced trait resulting from expression of stably-integrated,recombinant DNA for expressing a protein selected from the groupconsisting of SEQ ID NO: 5-8. More specifically the method comprises (a)screening a population of plants for an enhanced trait and a recombinantDNA, where individual plants in the population can exhibit the trait ata level less than, essentially the same as or greater than the levelthat the trait is exhibited in control plants which do not express therecombinant DNA, (b) selecting from the population one or more plantsthat exhibit the trait at a level greater than the level that said traitis exhibited in control plants, (c) verifying that the recombinant DNAis stably integrated in said selected plants, (d) analyzing tissue of aselected plant to determine the production of a protein having thefunction of a protein encoded by nucleotides in a sequence of one of SEQID NO:1-4; and (e) collecting seed from a selected plant. In one aspectof the invention the plants in the population further comprise DNAexpressing a protein that provides tolerance to exposure to an herbicideapplied at levels that are lethal to wild type plant cells and theselecting is effected by treating the population with the herbicide,e.g. a glyphosate, dicamba, or glufosinate compound. In another aspectof the invention the plants are selected by identifying plants with theenhanced trait. The methods are especially useful for manufacturingcorn, soybean, cotton, alfalfa, wheat or rice seed. In a another aspect,the plants further comprise a DNA expressing a second protein thatprovides plant cells with one or more enhanced agronomic traits.

Another aspect of the invention provides a method of producing hybridcorn seed comprising acquiring hybrid corn seed from a herbicidetolerant corn plant which also has stably-integrated, recombinant DNAcomprising a promoter that is (a) functional in plant cells and (b) isoperably linked to DNA that encodes a protein selected from the groupconsisting of SEQ ID NO: 5-8; wherein a progeny transgenic plantregenerated from a copy of said cell exhibits an enhanced trait ascompared to a control plant without said DNA construct; and wherein saidcell is selected from a population of cells transformed with said DNAconstruct by screening progeny plants of cells in said population for anenhanced trait as compared to said control plant, and wherein saidenhanced trait is selected from the group consisting of enhanced wateruse efficiency, enhanced cold tolerance, increased yield, enhancednitrogen use efficiency, enhanced seed protein and enhanced seed oilresulting from expression of said protein. The methods further compriseproducing corn plants from said hybrid corn seed, wherein a fraction ofthe plants produced from said hybrid corn seed is homozygous for saidrecombinant DNA, a fraction of the plants produced from said hybrid cornseed is hemizygous for said recombinant DNA, and a fraction of theplants produced from said hybrid corn seed has none of said recombinantDNA; selecting corn plants which are homozygous and hemizygous for saidrecombiant DNA by treating with an herbicide; collecting seed fromherbicide-treated-surviving corn plants and planting said seed toproduce further progeny corn plants; repeating the selecting andcollecting steps at least once to produce an inbred corn line; andcrossing the inbred corn line with a second corn line to produce hybridseed.

Another aspect of the invention provides a method of selecting a plantcomprising plant cells of the invention by using an immunoreactiveantibody to detect the presence of protein expressed by recombinant DNAin seed or plant tissue. Yet another aspect of the invention providesanti-counterfeit milled seed having, as an indication of origin, a plantcell of this invention.

Still other aspects of this invention relate to transgenic plants withenhanced water use efficiency or enhanced nitrogen use efficiency. Forinstance, this invention provides methods of growing a corn, cotton orsoybean crop without irrigation water comprising planting seed havingplant cells of the invention which are selected for enhanced water useefficiency. Alternatively methods comprise applying reduced irrigationwater, e.g. providing up to 300 millimeters of ground water during theproduction of a corn crop. This invention also provides methods ofgrowing a corn, cotton or soybean crop without added nitrogen fertilizercomprising planting seed having plant cells of the invention which areselected for enhanced nitrogen use efficiency.

DETAILED DESCRIPTION OF THE INVENTION

As used herein a “plant cell” means a plant cell that is transformedwith stably-integrated, non-natural, recombinant DNA, e.g. byAgrobacterium-mediated transformation or by bombardment usingmicroparticles coated with recombinant DNA or other means. A plant cellof this invention can be an originally-transformed plant cell thatexists as a microorganism or as a progeny plant cell that is regeneratedinto differentiated tissue, e.g. into a transgenic plant withstably-integrated, non-natural recombinant DNA, or seed or pollenderived from a progeny transgenic plant.

As used herein a “transgenic plant” means a plant whose genome has beenaltered by the stable integration of recombinant DNA. A transgenic plantincludes a plant regenerated from an originally-transformed plant celland progeny transgenic plants from later generations or crosses of atransformed plant.

As used herein “recombinant DNA” means DNA which has been a geneticallyengineered and constructed outside of a cell including DNA containingnaturally occurring DNA or cDNA or synthetic DNA.

As used herein “consensus sequence” means an artificial sequence ofamino acids in a conserved region of an alignment of amino acidsequences of homologous proteins, e.g. as determined by a CLUSTALWalignment of amino acid sequence of homolog proteins.

As used herein “homolog” means a protein in a group of proteins thatperform the same biological function, e.g. proteins that belong to thesame Pfam protein family and that provide a common enhanced trait intransgenic plants of this invention. Homologs are expressed byhomologous genes. Homologous genes include naturally occurring allelesand artificially-created variants. Degeneracy of the genetic codeprovides the possibility to substitute at least one base of the proteinencoding sequence of a gene with a different base without causing theamino acid sequence of the polypeptide produced from the gene to bechanged. Hence, a polynucleotide useful in the present invention mayhave any base sequence that has been changed from SEQ ID NO:1 throughSEQ ID NO:4 by substitution in accordance with degeneracy of the geneticcode. Homologs are proteins that, when optimally aligned, have at least60% identity, more preferably about 70% or higher, more preferably atleast 80% and even more preferably at least 90% identity over the fulllength of a protein identified as being associated with imparting anenhanced trait when expressed in plant cells. Homologs include proteinswith an amino acid sequence that has at least 90% identity to aconsensus amino acid sequence of proteins and homologs disclosed herein.

Homologs are be identified by comparison of amino acid sequence, e.g.manually or by use of a computer-based tool using known homology-basedsearch algorithms such as those commonly known and referred to as BLAST,FASTA, and Smith-Waterman. A local sequence alignment program, e.g.BLAST, can be used to search a database of sequences to find similarsequences, and the summary Expectation value (E-value) used to measurethe sequence base similarity. As a protein hit with the best E-value fora particular organism may not necessarily be an ortholog or the onlyortholog, a reciprocal query is used in the present invention to filterhit sequences with significant E-values for ortholog identification. Thereciprocal query entails search of the significant hits against adatabase of amino acid sequences from the base organism that are similarto the sequence of the query protein. A hit is a likely ortholog, whenthe reciprocal query's best hit is the query protein itself or a proteinencoded by a duplicated gene after speciation. A further aspect of theinvention comprises functional homolog proteins that differ in one ormore amino acids from those of disclosed protein as the result ofconservative amino acid substitutions, for example substitutions areamong: acidic (negatively charged) amino acids such as aspartic acid andglutamic acid; basic (positively charged) amino acids such as arginine,histidine, and lysine; neutral polar amino acids such as glycine,serine, threonine, cysteine, tyrosine, asparagine, and glutamine;neutral nonpolar (hydrophobic) amino acids such as alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan, and methionine;amino acids having aliphatic side chains such as glycine, alanine,valine, leucine, and isoleucine; amino acids having aliphatic-hydroxylside chains such as serine and threonine; amino acids havingamide-containing side chains such as asparagine and glutamine; aminoacids having aromatic side chains such as phenylalanine, tyrosine, andtryptophan; amino acids having basic side chains such as lysine,arginine, and histidine; amino acids having sulfur-containing sidechains such as cysteine and methionine; naturally conservative aminoacids such as valine-leucine, valine-isoleucine, phenylalanine-tyrosine,lysine-arginine, alanine-valine, aspartic acid-glutamic acid, andasparagine-glutamine. A further aspect of the homologs encoded by DNAuseful in the transgenic plants of the invention are those proteins thatdiffer from a disclosed protein as the result of deletion or insertionof one or more amino acids in a native sequence.

As used herein, “percent identity” means the extent to which twooptimally aligned DNA or protein segments are invariant throughout awindow of alignment of components, for example nucleotide sequence oramino acid sequence. An “identity fraction” for aligned segments of atest sequence and a reference sequence is the number of identicalcomponents that are shared by sequences of the two aligned segmentsdivided by the total number of sequence components in the referencesegment over a window of alignment which is the smaller of the full testsequence or the full reference sequence. “Percent identity” (“%identity”) is the identity fraction times 100.

As used herein “Pfam” refers to a large collection of multiple sequencealignments and hidden Markov models covering many common proteinfamilies, e.g. Pfam version 18.0 (August 2005) contains alignments andmodels for 7973 protein families and is based on the Swissprot 47.0 andSP-TrEMBL 30.0 protein sequence databases. See S. R. Eddy, “ProfileHidden Markov Models”, Bioinformatics 14:755-763, 1998. Pfam iscurrently maintained and updated by a Pfam Consortium. The alignmentsrepresent some evolutionary conserved structure that has implicationsfor the protein's function. Profile hidden Markov models (profile HMMs)built from the Pfam alignments are useful for automatically recognizingthat a new protein belongs to an existing protein family even if thehomology by alignment appears to be low. Once one DNA is identified asencoding a protein which imparts an enhanced trait when expressed intransgenic plants, other DNA encoding proteins in the same proteinfamily are identified by querying the amino acid sequence of proteinencoded by candidate DNA against the Hidden Markov Model whichcharacterizes the Pfam domain using HMMER software, a current version ofwhich is provided in the appended computer listing. Candidate proteinsmeeting the gathering cutoff for the alignment of a particular Pfam arein the protein family and have cognate DNA that is useful inconstructing recombinant DNA for the use in the plant cells of thisinvention. Hidden Markov Model databases for use with HMMER software inidentifying DNA expressing protein in a common Pfam for recombinant DNAin the plant cells of this invention are also included in the appendedcomputer listing. The HMMER software and Pfam databases are version 18.0and were used to determine that the amino acid sequence of SEQ ID NO:5is characterized by two Pfam domains, i.e. Homeobox domain and HALZdomain. The Homeobox domain was identified as comprising amino acidresidues between positions 130 and 193 with a score of 70.1 exceedingthe gathering cutoff of −4. The HALZ domain was identified as comprisingamino acid residues between positions 194 and 238 with a score of 71.9exceeding the gathering cutoff of 17.

The HMMER software and databases for identifying the Homeobox and HALZdomains are accessed at any Pfam website and can be provided by theapplicant, e.g. in an appended computer listing.

As used herein “promoter” means regulatory DNA for initializingtranscription. A “plant promoter” is a promoter capable of initiatingtranscription in plant cells whether or not its origin is a plant cell,e.g. is it well known that Agrobacterium promoters are functional inplant cells. Thus, plant promoters include promoter DNA obtained fromplants, plant viruses and bacteria such as Agrobacterium andBradyrhizobium bacteria. Examples of promoters under developmentalcontrol include promoters that preferentially initiate transcription incertain tissues, such as leaves, roots, or seeds. Such promoters arereferred to as “tissue preferred”. Promoters that initiate transcriptiononly in certain tissues are referred to as “tissue specific”. A “celltype” specific promoter primarily drives expression in certain celltypes in one or more organs, for example, vascular cells in roots orleaves. An “inducible” or “repressible” promoter is a promoter which isunder environmental control. Examples of environmental conditions thatmay effect transcription by inducible promoters include anaerobicconditions, or certain chemicals, or the presence of light. Tissuespecific, tissue preferred, cell type specific, and inducible promotersconstitute the class of “non-constitutive” promoters. A “constitutive”promoter is a promoter which is active under most conditions.

As used herein “operably linked” means the association of two or moreDNA fragments in a DNA construct so that the function of one, e.g.protein-encoding DNA, is controlled by the other, e.g. a promoter.

As used herein “expressed” means produced, e.g. a protein is expressedin a plant cell when its cognate DNA is transcribed to mRNA that istranslated to the protein.

As used herein a “control plant” means a plant that does not contain therecombinant DNA that expressed a protein that impart an enhanced trait.A control plant is to identify and select a transgenic plant that has anenhance trait. A suitable control plant can be a non-transgenic plant ofthe parental line used to generate a transgenic plant, i.e. devoid ofrecombinant DNA. A suitable control plant may in some cases be a progenyof a hemizygous transgenic plant line that is does not contain therecombinant DNA, known as a negative segregant.

As used herein an “enhanced trait” means a characteristic of atransgenic plant that includes, but is not limited to, an enhanceagronomic trait characterized by enhanced plant morphology, physiology,growth and development, yield, nutritional enhancement, disease or pestresistance, or environmental or chemical tolerance. In more specificaspects of this invention enhanced trait is selected from group ofenhanced traits consisting of enhanced water use efficiency, enhancedcold tolerance, increased yield, enhanced nitrogen use efficiency,enhanced seed protein and enhanced seed oil. In an important aspect ofthe invention the enhanced trait is enhanced yield including increasedyield under non-stress conditions and increased yield underenvironmental stress conditions. Stress conditions may include, forexample, drought, shade, fungal disease, viral disease, bacterialdisease, insect infestation, nematode infestation, cold temperatureexposure, heat exposure, osmotic stress, reduced nitrogen nutrientavailability, reduced phosphorus nutrient availability and high plantdensity. “Yield” can be affected by many properties including withoutlimitation, plant height, pod number, pod position on the plant, numberof internodes, incidence of pod shatter, grain size, efficiency ofnodulation and nitrogen fixation, efficiency of nutrient assimilation,resistance to biotic and abiotic stress, carbon assimilation, plantarchitecture, resistance to lodging, percent seed germination, seedlingvigor, and juvenile traits. Yield can also affected by efficiency ofgermination (including germination in stressed conditions), growth rate(including growth rate in stressed conditions), ear number, seed numberper ear, seed size, composition of seed (starch, oil, protein) andcharacteristics of seed fill.

Increased yield of a transgenic plant of the present invention can bemeasured in a number of ways, including test weight, seed number perplant, seed weight, seed number per unit area (i.e. seeds, or weight ofseeds, per acre), bushels per acre (bu/a), tonnes per acre, tons peracre, kilo per hectare. For example, maize yield may be measured asproduction of shelled corn kernels per unit of production area, forexample in bushels per acre or metric tons per hectare, often reportedon a moisture adjusted basis, for example at 15.5 percent moisture.Increased yield may result from improved utilization of key biochemicalcompounds, such as nitrogen, phosphorous and carbohydrate, or fromimproved responses to environmental stresses, such as cold, heat,drought, salt, and attack by pests or pathogens. Recombinant DNA used inthis invention can also be used to provide plants having improved growthand development, and ultimately increased yield, as the result ofmodified expression of plant growth regulators or modification of cellcycle or photosynthesis pathways. Also of interest is the generation oftransgenic plants that demonstrate enhanced yield with respect to a seedcomponent that may or may not correspond to an increase in overall plantyield. Such properties include enhancements in seed oil, seed moleculessuch as tocopherol, protein and starch, or oil particular oil componentsas may be manifest by an alteration in the ratios of seed components.

A subset of the nucleic molecules of this invention includes fragmentsof the disclosed recombinant DNA consisting of oligonucleotides of atleast 15, preferably at least 16 or 17, more preferably at least 18 or19, and even more preferably at least 20 or more, consecutivenucleotides. Such oligonucleotides are fragments of the larger moleculeshaving a sequence selected from the group consisting of SEQ ID NO:1through SEQ ID NO:4, and find use, for example as probes and primers fordetection of the polynucleotides of the present invention.

DNA constructs are assembled using methods well known to persons ofordinary skill in the art and typically comprise a promoter operablylinked to DNA, the expression of which provides the enhanced agronomictrait. Other construct components may include additional regulatoryelements, such as 5′ leaders and introns for enhancing transcription, 3′untranslated regions (such as polyadenylation signals and sites), DNAfor transit or signal peptides.

Numerous promoters that are active in plant cells have been described inthe literature. These include promoters present in plant genomes as wellas promoters from other sources, including nopaline synthase (NOS)promoter and octopine synthase (OCS) promoters carried on tumor-inducingplasmids of Agrobacterium tumefaciens, caulimovirus promoters such asthe cauliflower mosaic virus. For instance, see U.S. Pat. Nos. 5,858,742and 5,322,938, which disclose versions of the constitutive promoterderived from cauliflower mosaic virus (CaMV35S), U.S. Pat. No.5,641,876, which discloses a rice actin promoter, U.S. PatentApplication Publication 2002/0192813A1, which discloses 5′, 3′ andintron elements useful in the design of effective plant expressionvectors, U.S. patent application Ser. No. 09/757,089, which discloses amaize chloroplast aldolase promoter, U.S. patent application Ser. No.08/706,946, which discloses a rice glutelin promoter, U.S. patentapplication Ser. No. 09/757,089, which discloses a maize aldolase (FDA)promoter, and U.S. patent application Ser. No. 60/310, 370, whichdiscloses a maize nicotianamine synthase promoter, all of which areincorporated herein by reference. These and numerous other promotersthat function in plant cells are known to those skilled in the art andavailable for use in recombinant polynucleotides of the presentinvention to provide for expression of desired genes in transgenic plantcells.

In other aspects of the invention, preferential expression in plantgreen tissues is desired. Promoters of interest for such uses includethose from genes such as Arabidopsis thaliana ribulose-1,5-bisphosphatecarboxylase (Rubisco) small subunit (Fischhoff et al. (1992) Plant Mol.Biol. 20:81-93), aldolase and pyruvate orthophosphate dikinase (PPDK)(Taniguchi et al. (2000) Plant Cell Physiol. 41(1):42-48).

Furthermore, the promoters may be altered to contain multiple “enhancersequences” to assist in elevating gene expression. Such enhancers areknown in the art. By including an enhancer sequence with suchconstructs, the expression of the selected protein may be enhanced.These enhancers often are found 5′ to the start of transcription in apromoter that functions in eukaryotic cells, but can often be insertedupstream (5′) or downstream (3′) to the coding sequence. In someinstances, these 5′ enhancing elements are introns. Particularly usefulas enhancers are the 5′ introns of the rice actin 1 (see U.S. Pat. No.5,641,876) and rice actin 2 genes, the maize alcohol dehydrogenase geneintron, the maize heat shock protein 70 gene intron (U.S. Pat. No.5,593,874) and the maize shrunken 1 gene.

In other aspects of the invention, sufficient expression in plant seedtissues is desired to effect improvements in seed composition. Exemplarypromoters for use for seed composition modification include promotersfrom seed genes such as napin (U.S. Pat. No. 5,420,034), maize L3oleosin (U.S. Pat. No. 6,433,252), zein Z27 (Russell et al. (1997)Transgenic Res. 6(2):157-166), globulin 1 (Belanger et al (1991)Genetics 129:863-872), glutelin 1 (Russell (1997) supra), andperoxiredoxin antioxidant (Peri) (Stacy et al. (1996) Plant Mol Biol.31(6):1205-1216).

Recombinant DNA constructs prepared in accordance with the inventionwill also generally include a 3′ element that typically contains apolyadenylation signal and site. Well-known 3′ elements include thosefrom Agrobacterium tumefaciens genes such as nos 3′, tml 3′, tmr 3′, tms3′, ocs 3′, tr7 3′, for example disclosed in U.S. Pat. No. 6,090,627,incorporated herein by reference; 3′ elements from plant genes such aswheat (Triticum aesevitum) heat shock protein 17 (Hsp17 3), a wheatubiquitin gene, a wheat fructose-1,6-biphosphatase gene, a rice glutelingene a rice lactate dehydrogenase gene and a rice beta-tubulin gene, allof which are disclosed in U.S. published patent application 2002/0192813A1, incorporated herein by reference; and the pea (Pisum sativum)ribulose biphosphate carboxylase gene (rbs 3 ‘), and 3’ elements fromthe genes within the host plant.

Constructs and vectors may also include a transit peptide for targetingof a gene target to a plant organelle, particularly to a chloroplast,leucoplast or other plastid organelle. For descriptions of the use ofchloroplast transit peptides see U.S. Pat. No. 5,188,642 and U.S. Pat.No. 5,728,925, incorporated herein by reference. For description of thetransit peptide region of an Arabidopsis EPSPS gene useful in thepresent invention, see Klee, H. J. et al (MGG (1987) 210:437-442).

Transgenic plants comprising or derived from plant cells of thisinvention transformed with recombinant DNA can be further enhanced withstacked traits, e.g. a crop plant having an enhanced trait resultingfrom expression of DNA disclosed herein in combination with herbicideand/or pest resistance traits. For example, genes of the currentinvention can be stacked with other traits of agronomic interest, suchas a trait providing herbicide resistance, or insect resistance, such asusing a gene from Bacillus thuringensis to provide resistance againstlepidopteran, coliopteran, homopteran, hemiopteran, and other insects.Herbicides for which transgenic plant tolerance has been demonstratedand the method of the present invention can be applied include, but arenot limited to, glyphosate, dicamba, glufosinate, sulfonylurea,bromoxynil and norflurazon herbicides. Polynucleotide molecules encodingproteins involved in herbicide tolerance are well-known in the art andinclude, but are not limited to, a polynucleotide molecule encoding5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) disclosed in U.S.Pat. Nos. 5,094,945; 5,627,061; 5,633,435 and 6,040,497 for impartingglyphosate tolerance; polynucleotide molecules encoding a glyphosateoxidoreductase (GOX) disclosed in U.S. Pat. No. 5,463,175 and aglyphosate-N-acetyl transferase (GAT) disclosed in U.S. PatentApplication publication 2003/0083480 A1 also for imparting glyphosatetolerance; dicamba monooxygenase disclosed in U.S. Patent Applicationpublication 2003/0135879 A1 for imparting dicamba tolerance; apolynucleotide molecule encoding bromoxynil nitrilase (Bxn) disclosed inU.S. Pat. No. 4,810,648 for imparting bromoxynil tolerance; apolynucleotide molecule encoding phytoene desaturase (crtl) described inMisawa et al, (1993) Plant J. 4:833-840 and Misawa et al, (1994) PlantJ. 6:481-489 for norflurazon tolerance; a polynucleotide moleculeencoding acetohydroxyacid synthase (AHAS, aka ALS) described inSathasiivan et al. (1990) Nucl. Acids Res. 18:2188-2193 for impartingtolerance to sulfonylurea herbicides; polynucleotide molecules known asbar genes disclosed in DeBlock, et al. (1987) EMBO J. 6:2513-2519 forimparting glufosinate and bialaphos tolerance; polynucleotide moleculesdisclosed in U.S. Patent Application Publication 2003/010609 A1 forimparting N-amino methyl phosphonic acid tolerance; polynucleotidemolecules disclosed in U.S. Pat. No. 6,107,549 for imparting pyridineherbicide resistance; molecules and methods for imparting tolerance tomultiple herbicides such as glyphosate, atrazine, ALS inhibitors,isoxoflutole and glufosinate herbicides are disclosed in U.S. Pat. No.6,376,754 and U.S. Patent Application Publication 2002/0112260, all ofsaid U.S. patents and patent application Publications are incorporatedherein by reference. Molecules and methods for impartinginsect/nematode/virus resistance are disclosed in U.S. Pat. Nos.5,250,515; 5,880,275; 6,506,599; 5,986,175 and U.S. Patent ApplicationPublication 2003/0150017 A1, all of which are incorporated herein byreference.

In particular embodiments, the inventors contemplate the use ofantibodies, either monoclonal or polyclonal which bind to the proteinsdisclosed herein. Means for preparing and characterizing antibodies arewell known in the art (See, e.g., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988; incorporated herein by reference). Themethods for generating monoclonal antibodies (mAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogenic composition in accordance with the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically the animalused for production of anti-antisera is a rabbit, a mouse, a rat, ahamster, a guinea pig or a goat. Because of the relatively large bloodvolume of rabbits, a rabbit is a preferred choice for production ofpolyclonal antibodies.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include using glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

mAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference.

Typically, this technique involves immunizing a suitable animal with aselected immunogen composition, e.g., a purified or partially purifiedantifungal protein, polypeptide or peptide. The immunizing compositionis administered in a manner effective to stimulate antibody producingcells. Rodents such as mice and rats are preferred animals, however, theuse of rabbit, sheep, or frog cells is also possible. The use of ratsmay provide certain advantages (Goding, 1986, pp. 60-61), but mice arepreferred, with the BALB/c mouse being most preferred as this is mostroutinely used and generally gives a higher percentage of stablefusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render them incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, 1986, pp. 65-66; Campbell, 1984, pp.75-83). For example, where the immunized animal is a mouse, one may useP3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11,MPC11-X45-GTG 1.7 and S194/5XXO Bul; for rats, one may use R210.RCY3,Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 andUC729-6 are all useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Spend virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, (Gefter et al., 1977). The use of electrically inducedfusion methods is also appropriate (Goding, 1986, pp. 71-74).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azasenne blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

Plant Cell Transformation Methods

Numerous methods for transforming plant cells with recombinant DNA areknown in the art and may be used in the present invention. Two commonlyused methods for plant transformation are Agrobacterium-mediatedtransformation and microprojectile bombardment. Microprojectilebombardment methods are illustrated in U.S. Pat. No. 5,015,580(soybean); U.S. Pat. No. 5,550,318 (corn); U.S. Pat. No. 5,538,880(corn); U.S. Pat. No. 5,914,451 (soybean); U.S. Pat. No. 6,160,208(corn); U.S. Pat. No. 6,399,861 (corn) and U.S. Pat. No. 6,153,812(wheat) and Agrobacterium-mediated transformation is described in U.S.Pat. No. 5,159,135 (cotton); U.S. Pat. No. 5,824,877 (soybean); U.S.Pat. No. 5,591,616 (corn); and U.S. Pat. No. 6,384,301 (soybean), all ofwhich are incorporated herein by reference. For Agrobacteriumtumefaciens based plant transformation system, additional elementspresent on transformation constructs will include T-DNA left and rightborder sequences to facilitate incorporation of the recombinantpolynucleotide into the plant genome.

In general it is useful to introduce recombinant DNA randomly, i.e. at anon-specific location, in the genome of a target plant line. In specialcases it may be useful to target recombinant DNA insertion in order toachieve site-specific integration, for example to replace an existinggene in the genome, to use an existing promoter in the plant genome, orto insert a recombinant polynucleotide at a predetermined site known tobe active for gene expression. Several site specific recombinationsystems exist which are known to function implants include cre-lox asdisclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S.Pat. No. 5,527,695, both incorporated herein by reference.

Transformation methods of this invention are preferably practiced intissue culture on media and in a controlled environment. “Media” refersto the numerous nutrient mixtures that are used to grow cells in vitro,that is, outside of the intact living organism. Recipient cell targetsinclude, but are not limited to, meristem cells, callus, immatureembryos and gametic cells such as microspores, pollen, sperm and eggcells. It is contemplated that any cell from which a fertile plant maybe regenerated is useful as a recipient cell. Callus may be initiatedfrom tissue sources including, but not limited to, immature embryos,seedling apical meristems, microspores and the like. Cells capable ofproliferating as callus are also recipient cells for genetictransformation. Practical transformation methods and materials formaking transgenic plants of this invention, for example various mediaand recipient target cells, transformation of immature embryo cells andsubsequent regeneration of fertile transgenic plants are disclosed inU.S. Pat. Nos. 6,194,636 and 6,232,526, which are incorporated herein byreference.

The seeds of transgenic plants can be harvested from fertile transgenicplants and be used to grow progeny generations of transformed plants ofthis invention including hybrid plants line for selection of plantshaving an enhanced trait. In addition to direct transformation of aplant with a recombinant DNA, transgenic plants can be prepared bycrossing a first plant having a recombinant DNA with a second plantlacking the DNA. For example, recombinant DNA can be introduced intofirst plant line that is amenable to transformation to produce atransgenic plant which can be crossed with a second plant line tointrogress the recombinant DNA into the second plant line. A transgenicplant with recombinant DNA providing an enhanced trait, e.g. enhancedyield, can be crossed with transgenic plant line having otherrecombinant DNA that confers another trait, for example herbicideresistance or pest resistance, to produce progeny plants havingrecombinant DNA that confers both traits. Typically, in such breedingfor combining traits the transgenic plant donating the additional traitis a male line and the transgenic plant carrying the base traits is thefemale line. The progeny of this cross will segregate such that some ofthe plants will carry the DNA for both parental traits and some willcarry DNA for one parental trait; such plants can be identified bymarkers associated with parental recombinant DNA, e.g. markeridentification by analysis for recombinant DNA or, in the case where aselectable marker is linked to the recombinant, by application of theselecting agent such as a herbicide for use with a herbicide tolerancemarker, or by selection for the enhanced trait. Progeny plants carryingDNA for both parental traits can be crossed back into the female parentline multiple times, for example usually 6 to 8 generations, to producea progeny plant with substantially the same genotype as one originaltransgenic parental line but for the recombinant DNA of the othertransgenic parental line

In the practice of transformation DNA is typically introduced into onlya small percentage of target plant cells in any one transformationexperiment. Marker genes are used to provide an efficient system foridentification of those cells that are stably transformed by receivingand integrating a transgenic DNA construct into their genomes. Preferredmarker genes provide selective markers which confer resistance to aselective agent, such as an antibiotic or herbicide. Any of theherbicides to which plants of this invention may be resistant are usefulagents for selective markers. Potentially transformed cells are exposedto the selective agent. In the population of surviving cells will bethose cells where, generally, the resistance-conferring gene isintegrated and expressed at sufficient levels to permit cell survival.Cells may be tested further to confirm stable integration of theexogenous DNA. Commonly used selective marker genes include thoseconferring resistance to antibiotics such as kanamycin and paromomycin(nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) orresistance to herbicides such as glufosinate (bar or pat) and glyphosate(aroA or EPSPS). Examples of such selectable are illustrated in U.S.Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047, all of whichare incorporated herein by reference. Selectable markers which providean ability to visually identify transformants can also be employed, forexample, a gene expressing a colored or fluorescent protein such as aluciferase or green fluorescent protein (GFP) or a gene expressing abeta-glucuronidase or uidA gene (GUS) for which various chromogenicsubstrates are known.

Plant cells that survive exposure to the selective agent, or plant cellsthat have been scored positive in a screening assay, may be cultured inregeneration media and allowed to mature into plants. Developingplantlets regenerated from transformed plant cells can be transferred toplant growth mix, and hardened off, for example, in an environmentallycontrolled chamber at about 85% relative humidity, 600 ppm CO₂, and25-250 microeinsteins m⁻² s⁻¹ of light, prior to transfer to agreenhouse or growth chamber for maturation. Plants are regenerated fromabout 6 weeks to 10 months after a transformant is identified, dependingon the initial tissue. Plants may be pollinated using conventional plantbreeding methods known to those of skill in the art and seed produced,for example self-pollination is commonly used with transgenic corn. Theregenerated transformed plant or its progeny seed or plants can betested for expression of the recombinant DNA and selected for thepresence of enhanced agronomic trait.

Transgenic Plants and Seeds

Transgenic plants derived from the plant cells of this invention aregrown to generate transgenic plants having an enhanced trait as comparedto a control plant and produce transgenic seed and haploid pollen ofthis invention. Such plants with enhanced traits are identified byselection of transformed plants or progeny seed for the enhanced trait.For efficiency a selection method is designed to evaluate multipletransgenic plants (events) comprising the recombinant DNA, for examplemultiple plants from 2 to 20 or more transgenic events. Transgenicplants grown from transgenic seed provided herein demonstrate improvedagronomic traits that contribute to increased yield or other trait thatprovides increased plant value, including, for example, improved seedquality. Of particular interest are plants having enhanced water useefficiency, enhanced cold tolerance, increased yield, enhanced nitrogenuse efficiency, enhanced seed protein and enhanced seed oil.

Table 1 provides a list of protein encoding DNA (“genes”) that areuseful as recombinant DNA for production of transgenic plants withenhanced agronomic trait, the elements of Table 1 are described byreference to:

“PEP SEQ” which identifies an amino acid sequence from SEQ ID NO:5-8.“NUC SEQ” which identifies a DNA sequence from SEQ ID NO:1-4.“Base Vector” which identifies a base plasmid used for transformation ofthe recombinant DNA.“PROTEIN NAME” which is a common name for protein encoded by therecombinant DNA.“Plasmid ID” which identifies an arbitrary name for the planttransformation plasmid comprising recombinant DNA for expressing therecombinant DNA in plant cells.

TABLE 1 PEP NUC SEQ SEQ ID NO ID NO Base Vector PROTEIN NAME Plasmid ID5 1 pMON65154 Arabidopsis G1543 pMON68392 5 1 Arabidopsis G1543pMON74775 5 1 pMON74537 Arabidopsis G1543 pMON83062 6 2 pMON81244 CornG1543-like 1 pMON82686 6 2 pMON74537 Corn G1543-like 1 pMON83049 7 3pMON81244 Soy G1543-like 1 pMON82688 7 3 pMON81244 Soy G1543-like 1pMON84131 7 3 pMON74537 Soy G1543-like 1 pMON83311 8 4 pMON74537 riceHox3 - AAD37696 pMON73829Screening Methods for Transgenic Plants with Enhanced Agronomic Trait

Many transgenic events which survive to fertile transgenic plants thatproduce seeds and progeny plants will not exhibit an enhanced agronomictrait. Screening is necessary to identify the transgenic plant of thisinvention. Transgenic plants having enhanced agronomic traits areidentified from populations of plants transformed as described herein byevaluating the trait in a variety of assays to detect an enhancedagronomic trait. These assays also may take many forms, including butnot limited to, analyses to detect changes in the chemical composition,biomass, physiological properties, morphology of the plant. Changes inchemical compositions such as nutritional composition of grain can bedetected by analysis of the seed composition and content of protein,free amino acids, oil, free fatty acids, starch or tocopherols. Changesin biomass characteristics can be made on greenhouse or field grownplants and can include plant height, stem diameter, root and shoot dryweights; and, for corn plants, ear length and diameter. Changes inphysiological properties can be identified by evaluating responses tostress conditions, e.g., assays using imposed stress conditions such aswater deficit, nitrogen deficiency, cold growing conditions, pathogen orinsect attack or light deficiency, or increased plant density. Changesin morphology can be measured by visual observation of tendency of atransformed plant with an enhanced agronomic trait to also appear to bea normal plant as compared to changes toward bushy, taller, thicker,narrower leaves, striped leaves, knotted trait, chlorosis, albino,anthocyanin production, or altered tassels, ears or roots. Otherscreening properties include days to pollen shed, days to silking, leafextension rate, chlorophyll content, leaf temperature, stand, seedlingvigor, internode length, plant height, leaf number, leaf area,tillering, brace roots, stay green, stalk lodging, root lodging, planthealth, barreness/prolificacy, green snap, and pest resistance. Inaddition, phenotypic characteristics of harvested grain may beevaluated, including number of kernels per row on the ear, number ofrows of kernels on the ear, kernel abortion, kernel weight, kernel size,kernel density and physical grain quality.

Although preferred seeds for transgenic plants with enhanced agronomictraits of this invention are corn and soybean plants, other seeds arefor cotton, canola, wheat, sunflower, sorghum, alfalfa, barley, millet,rice, tobacco, fruit and vegetable crops, and turfgrass.

Screening for Enhanced Nitrogen Use Efficiency

One preferred enhanced agronomic trait in transgenic plants of thisinvention is enhanced nitrogen use efficiency as compared to controlplants. Higher nitrogen soil applications increase seed protein andstarch accumulation, and lead to larger seed weight and larger kernelnumber per ear. Recent improvements in elite high yielding corn hybridgenotypes include the ability to utilize nitrogen efficiently. Genescausing the enhanced nitrogen use efficiency in crop plants areespecially useful, e.g., for improving yield. Enhanced nitrogen useefficiency can be assessed by measuring changes in plant growth such asleaf area production, shoot biomass, chlorophyll content in plants grownin nitrogen limiting conditions and/or nitrogen sufficient conditions.It is useful to conduct a first screen in nitrogen limiting conditionsand confirm replicate transgenic events in both nitrogen limiting andnitrogen sufficient conditions. Table 2 shows the amount of nutrients inthe nutrient solution for nitrogen limiting conditions (low nitrogengrowth condition) and nitrogen sufficient conditions (high nitrogengrowth condition) useful for nitrogen use efficiency screening. Forexample in a greenhouse screen pots of transgenic plants and controlplants are treated with 100 ml of nutrient solution three times a weekon alternate days starting at 8 and 10 days after planting for highnitrogen and low nitrogen screening, respectively.

TABLE 2 2 mM NH₄NO₃ (low 20 mM NH₄NO₃ Nitrogen growth (high Nitrogengrowth condition) condition) Nutrient Stock mL/L mL/L 1M NH₄N0₃ 2 20 1MKH₂PO₄ 0.5 0.5 1M MgSO₄•7H₂O 2 2 1M CaCl₂ 2.5 2.5 1M K₂SO₄ 1 1 Note:Adjust pH to 5.6 with HCl or KOH

After 28 days of plant growth for low nitrogen screening and 23 days forhigh nitrogen screening, measurements are taken for: total shoot freshmass, leaf chlorophyll, leaf area, leaf fresh mass and leaf dry mass.

Screening for Increased Yield

Many transgenic plants of this invention exhibit enhanced yield ascompared to a control plant. Enhanced yield can result from enhancedseed sink potential, i.e. the number and size of endosperm cells orkernels and/or enhanced sink strength, i.e. the rate of starchbiosynthesis. Sink potential can be established very early during kerneldevelopment, as endosperm cell number and cell size are determinedwithin the first few days after pollination.

Much of the increase in corn yield of the past several decades hasresulted from an increase in planting density. During that period, cornyield has been increasing at a rate of 2.1 bushels/acre/year, but theplanting density has increased at a rate of 250 plants/acre/year. Acharacteristic of modern hybrid corn is the ability of these varietiesto be planted at high density. Many studies have shown that a higherthan current planting density should result in more biomass production,but current germplasm does not perform well at these higher densities.One approach to increasing yield is to increase harvest index (HI), theproportion of biomass that is allocated to the kernel compared to totalbiomass, in high density plantings.

Effective yield screening of transgenic corn uses hybrid progeny of thetransgenic event over multiple locations with plants grown under optimalproduction management practices, and maximum pest control. A usefultarget for enhanced yield is a 5% to 10% increase in yield as comparedto yield produced by plants grown from seed for a control plant. Usefulscreening in multiple and diverse geographic locations, e.g., up to 16or more locations, over one or more plating seasons, e.g., at least twoplanting seasons to statistically distinguish yield improvement fromnatural environmental effects. It is to plant multiple transgenicplants, positive and negative control plants, and pollinator plants instandard plots, e.g., 2 row plots, 20 feet long by 5 feet wide with 30inches distance between rows and a 3 foot alley between ranges.Transgenic events can be grouped by recombinant DNA constructs withgroups randomly placed in the field. A pollinator plot of a high qualitycorn line is planted for every two plots to allow open pollination whenusing male sterile transgenic events. A useful planting density is about30,000 plants/acre.

Surrogate indicators for screening for yield improvement include sourcecapacity (biomass), source output (sucrose and photosynthesis), sinkcomponents (kernel size, ear size, starch in the seed), development(light response, height, density tolerance), maturity, early floweringtrait and physiological responses to high density planting, e.g., at45,000 plants per acre, e.g., as illustrated in Table 3 and 4.

TABLE 3 Timing Evaluation Description comments V2-3 Early stand Can betaken any time after germination and prior to removal of any plants.Pollen shed GDU to 50% shed GDU to 50% plants shedding 50% tassel.Silking GDU to 50% silk GDU to 50% plants showing silks. Maturity Plantheight Height from soil surface to 10 plants per plot - Yield flag leafattachment (inches). team assistance Maturity Ear height Height fromsoil surface to 10 plants per plot - Yield primary ear attachment node.team assistance Maturity Leaves above ear visual scores: erect, size,rolling Maturity Tassel size Visual scores +/− vs. WT Pre-Harvest FinalStand Final stand count prior to harvest, exclude tillers Pre-HarvestStalk lodging No. of stalks broken below the primary ear attachment.Exclude leaning tillers Pre-Harvest Root lodging No. of stalksleaning >45° angle from perpendicular. Pre-Harvest Stay green Afterphysiological maturity and when differences among genotypes are evident:Scale 1 (90-100% tissue green) - 9 (0-19% tissue green). Harvest GrainYield Grain yield/plot (Shell weight)

When screening for yield improvement a useful statistical measurementapproach comprises three components, i.e. modeling spatialautocorrelation of the test field separately for each location,adjusting traits of recombinant DNA events for spatial dependence foreach location, and conducting an across location analysis. The firststep in modeling spatial autocorrelation is estimating the covarianceparameters of the semivariogram. A spherical covariance model is assumedto model the spatial autocorrelation. Because of the size and nature ofthe trial, it is likely that the spatial autocorrelation may change.Therefore, anisotropy is also assumed along with spherical covariancestructure. The following set of equations describes the statistical formof the anisotropic spherical covariance model.

${C\left( {h;\theta} \right)} = {{{vI}\left( {h = 0} \right)} + {{\sigma^{2}\left( {1 - {\frac{3}{2}h} + {\frac{1}{2}h^{3}}} \right)}{I\left( {h < 1} \right)}}}$

where I(•) is the indicator function h=√{square root over ({dot over(x)}²+{dot over (y)}²)}and

{dot over (x)}=[cos(ρπ/180)(x ₁ −x ₂)−sin(ρπ/180)(y ₁ −y ₂)]/ω_(x)

{dot over (y)}=[sin(ρπ/180)(x ₁ −x ₂)+cos(ρπ/180)(y ₁ −y ₂)]/ω_(y)

where s₁=(x₁, y₁) are the spatial coordinates of one location ands₂=(x₂, y₂) are the spatial coordinates of the second location. Thereare 5 covariance parameters,

θ=(ν,σ²,ρ,ω_(n),ω_(j))

where ν is the nugget effect, σ² is the partial sill, ρ is a rotation indegrees clockwise from north, ω_(n) is a scaling parameter for the minoraxis and ω_(j) is a scaling parameter for the major axis of ananisotropical ellipse of equal covariance. The five covarianceparameters that define the spatial trend will then be estimated by usingdata from heavily replicated pollinator plots via restricted maximumlikelihood approach. In a multi-location field trial, spatial trend aremodeled separately for each location.

After obtaining the variance parameters of the model, avariance-covariance structure is generated for the data set to beanalyzed. This variance-covariance structure contains spatialinformation required to adjust yield data for spatial dependence. Inthis case, a nested model that best represents the treatment andexperimental design of the study is used along with thevariance-covariance structure to adjust the yield data. During thisprocess the nursery or the seed batch effects can also be modeled andestimated to adjust the yields for any yield parity caused by seed batchdifferences.

After spatially adjusted data from different locations are generated,all adjusted data is combined and analyzed assuming locations asreplications. In this analysis, intra and inter-location variances arecombined to estimate the standard error of yield from transgenic plantsand control plants. Relative mean comparisons are used to indicatestatistically significant yield improvements.

Screening for Water Use Efficiency

An aspect of this invention provides transgenic plants with enhancedyield resulting from enhanced water use efficiency and/or droughttolerance. Described in this example is a high-throughput method forgreenhouse selection of transgenic corn plants to wild type corn plants(tested as inbreds or hybrids) for water use efficiency. This selectionprocess imposes 3 drought/re-water cycles on plants over a total periodof 15 days after an initial stress free growth period of 11 days. Eachcycle consists of 5 days, with no water being applied for the first fourdays and a water quenching on the 5th day of the cycle. The primaryphenotypes analyzed by the selection method are the changes in plantgrowth rate as determined by height and biomass during a vegetativedrought treatment. The hydration status of the shoot tissues followingthe drought is also measured. The plant heights are measured at threetime points. The first is taken just prior to the onset drought when theplant is 11 days old, which is the shoot initial height (SIH). The plantheight is also measured halfway throughout the drought/re-water regimen,on day 18 after planting, to give rise to the shoot mid-drought height(SMH). Upon the completion of the final drought cycle on day 26 afterplanting, the shoot portion of the plant is harvested and measured for afinal height, which is the shoot wilt height (SWH) and also measured forshoot wilted biomass (SWM). The shoot is placed in water at 40 degreeCelsius in the dark. Three days later, the shoot is weighted to giverise to the shoot turgid weight (STM). After drying in an oven for fourdays, the shoots are weighted for shoot dry biomass (SDM). The shootaverage height (SAH) is the mean plant height across the 3 heightmeasurements. The procedure described above may be adjusted for +/−˜oneday for each step given the situation.

To correct for slight differences between plants, a size correctedgrowth value is derived from SIH and SWH. This is the Relative GrowthRate (RGR). Relative Growth Rate (RGR) is calculated for each shootusing the formula [RGR % (SWH−SIH)/((SWH+SIH)/2)*100]. Relative watercontent (RWC) is a measurement of how much (%) of the plant was water atharvest. Water Content (RWC) is calculated for each shoot using theformula [RWC %=(SWM−SDM)/(STM−SDM)*100]. Fully watered corn plants ofthis age run around 98% RWC.

Screening for Growth Under Cold Stress

An aspect of this invention provides transgenic plants with enhancedgrowth under cold stress, e.g., in an early seedling growth assay. In anearly seedling growth assay 3 sets of seeds are assayed. The first setis a group of transgenic seeds from transgenic plants; the second set isnegative segregants of the transgenic seed; and the third seed set isseed from two cold tolerant and two cold sensitive wild-type controls.All seeds are treated with a fungicide as indicated above. Seeds aregrown in germination paper (12 inch×18 inch pieces of Anchor Paper#SD7606), wetted in a solution of 0.5% KNO3 and 0.1% Thyram. For eachpaper fifteen seeds are placed on the line evenly spaced such that theradical s will grow toward the same edge. The wet paper is rolled upevenly and tight enough to hold the seeds in place. The roll is securedinto place with two large paper clips, one at the top and one at thebottom. The rolls are incubated in a growth chamber at 23 degree C. forthree days in a randomized complete block design within an appropriatecontainer. The chamber is set for 65% humidity with no light cycle. Forthe cold stress treatment the rolls are then incubated in a growthchamber at 12 degree C. for fourteen days. The chamber is set for 65%humidity with no light cycle. For the warm treatment the rolls areincubated at 23 degree C. for an additional two days. After thetreatment the germination papers are unrolled and the seeds that did notgerminate are discarded. The lengths of the radicle and coleoptile foreach seed are measured. A coleoptile sample is collected from sixindividual kernels of each entry for confirming the expression ofrecombinant DNA. Statistical differences in the length of radical andshoot during pre-shock and cold shock are used for an estimation of theeffect of the cold treatment on corn plants. The analysis is conductedindependently for the warm and cold treatments.

Screen for Enhanced Oil, Starch, or Protein Levels in Plant Seeds

Oil levels of plant seeds are determined by low-resolution .sup.1Hnuclear magnetic resonance (NMR) (Tiwari et al., JAOCS, 51:104-109(1974); or Rubel, JAOCS, 71:1057-1062 (1994)). Alternatively, oil,starch and protein levels in seeds are determined by near infraredspectroscopy (NIR).

The following examples illustrate aspects of the invention.

Example 1

This example illustrates the construction of plasmids for transferringrecombinant DNA into plant cells which can be regenerated intotransgenic plants of this invention. Primers for PCR amplification ofprotein coding nucleotides of recombinant DNA were designed at or nearthe start and stop codons of the coding sequence, in order to eliminatemost of the 5′ and 3′ untranslated regions. Each recombinant DNA codingfor a protein identified in Table 1 was amplified by PCR prior toinsertion into the insertion site of one of the base vectors asreferenced in Table 1.

A base plant transformation vector pMON65154 was fabricated for use inpreparing recombinant DNA for transformation into corn tissue usingGATEWAY™ a Destination plant expression vector systems (available fromInvitrogen Life Technologies, Carlsbad, Calif.). With reference to theelements described in Table 5 below and SEQ ID NO:9, pMON65154 comprisesa selectable marker expression cassette and a template recombinant DNAexpression cassette. The marker expression cassette comprises a CaMV 35Spromoter operably linked to a gene encoding neomycin phosphotransferaseII (nptII) followed by a 3′ region of an Agrobacterium tumefaciensnopaline synthase gene (nos). The template recombinant DNA expressioncassette is positioned tail to tail with the marker expression cassette.The template recombinant DNA expression cassette comprises 5′ regulatoryDNA including a rice actin 1 promoter, exon and intron, followed by aGATEWAY™ insertion site for recombinant DNA, followed by a 3′ region ofa potato proteinase inhibitor II (pinII) gene. Once recombinant DNA hasbeen inserted into the insertion site, the plasmid is useful for planttransformation, for example by microprojectile bombardment.

TABLE 5 FUNCTION ELEMENT REFERENCE Plant gene of interest Rice actin 1promoter U.S. Pat. No. 5,641,876 expression cassette Rice actin 1 exon1, intron 1 U.S. Pat. No. 5,641,876 enhancer Gene of interest AttR1GATEWAY ™ Cloning Technology insertion site Instruction Manual CmR geneGATEWAY ™ Cloning Technology Instruction Manual ccdA, ccdB genesGATEWAY ™ Cloning Technology Instruction Manual attR2 GATEWAY ™ CloningTechnology Instruction Manual Plant gene of interest Potato pinII 3′region An et al. (1989) Plant Cell 1: 115-122 expression cassette Plantselectable CaMV 35S promoter U.S. Pat. No. 5,858,742 marker expressionnptII selectable marker U.S. Pat. No. 5,858,742 cassette nos 3′ regionU.S. Pat. No. 5,858,742 Maintenance in E. coli ColE1 origin ofreplication F1 origin of replication Bla ampicillin resistancesimilar base vector plasmid pMON72472 (SEQ ID NO: 10) was constructedfor use in Agrobacterium-mediated methods of plant transformationsimilar to pMON65154 except (a) the 5′ regulatory DNA in the templaterecombinant DNA expression cassette was a rice actin promoter and a riceactin intron, (b) left and right T-DNA border sequences fromAgrobacterium are added with the right border sequence is located 5′ tothe rice actin 1 promoter and the left border sequence is located 3′ tothe 35S promoter and (c) DNA is added to facilitate replication of theplasmid in both E. coli and Agrobacterium tumefaciens. The DNA added tothe plasmid outside of the T-DNA border sequences includes an oriV widehost range origin of DNA replication functional in Agrobacterium, apBR322 origin of replication functional in E. coli, and aspectinomycin/stretptomycin resistance gene for selection in both E.coli and Agrobacterium. pMON74775 is constructed in a base vectoressentially the same as pMON72472.

Other base vectors similar to those described above were alsoconstructed including pMON81244 containing a pyruvate orthophosphatedikinase (PPDK) promoter (SEQ ID NO: 11) and a maize DnaK intron (SEQ IDNO: 12) as an enhancer.

Plant Expression Vector for Soybean Transformation

Plasmids for use in transformation of soybean were also prepared.Elements of an exemplary common expression vector plasmid pMON74532 (SEQID NO:13) are shown in Table 7 below.

A plasmid vector similar to that described above for soy transformationwas constructed for use in Agrobacterium-mediated soybeantransformation, pMON74537, which contains the Arabidopsis thalianaribulose-1,5-bisphosphate carboxylase (Rubisco) small subunit promoter(SEQ ID NO: 14)

Protein coding segments of recombinant DNA are amplified by PCR prior toinsertion into vectors at the insertion site. Primers for PCRamplification are designed at or near the start and stop codons of thecoding sequence, in order to eliminate most of the 5′ and 3′untranslated regions.

TABLE 7 Function Element Reference Agro transformation B-ARGtu.rightborder Depicker, A. et al (1982) Mol Appl Genet 1: 561-573 Antibioticresistance CR-Ec.aadA-SPC/STR Repressor of primers from the ColE1CR-Ec.rop plasmid Origin of replication OR-Ec.oriV-RK2 Agrotransformation B-ARGtu.left border Barker, R. F. et al (1983) Plant MolBiol 2: 335-350 Plant selectable marker expression Promoter with intronand McDowell et al. (1996) cassette 5′UTR of Arabidopsis act 7 PlantPhysiol. 111: 699-711. gene (AtAct7) 5′ UTR of Arabidopsis act 7 geneIntron in 5′UTR of AtAct7 Transit peptide region of Klee, H. J. et al(1987) Arabidopsis EPSPS MGG 210: 437-442 Synthetic CP4 coding regionwith dicot preferred codon usage A 3′ UTR of the nopaline U.S. Pat. No.5,858,742 synthase gene of Agrobacterium tumefaciens Ti plasmid Plantgene of interest expression Promoter for 35S RNA from U.S. Pat. No.5,322,938 cassette CaMV containing a duplication of the −90 to −350region Gene of interest insertion site Cotton E6 3′ end GenBankaccession U30508

Example 2

This example illustrates plant transformation useful in producing thetransgenic corn plants of this invention. Corn plants of a readilytransformable line are grown in the greenhouse and ears harvested whenthe embryos are 1.5 to 2.0 mm in length. Ears are surface sterilized byspraying or soaking the ears in 80% ethanol, followed by air drying.Immature embryos are isolated from individual kernels on surfacesterilized ears. Prior to inoculation of maize cells, Agrobacteriumcells are grown overnight at room temperature. Immature maize embryosare inoculated with Agrobacterium shortly after excision, and incubatedat room temperature with Agrobacterium for 5-20 minutes. Immatureembryos are then co-cultured with Agrobacterium for 1 to 3 days at 23°C. in the dark. Co-cultured embryos are transferred to selection mediaand cultured for approximately two weeks to allow embryogenic callus todevelop. Embryogenic callus is transferred to culture medium containing100 mg/L paromomycin and subcultured at about two week intervals.Transformants are recovered 6 to 8 weeks after initiation of selection.

Plasmid vectors are prepared cloning DNA identified in Table 1 in theidentified base for use in corn transformation to produce transgeniccorn plants and seed.

For Agrobacterium-mediated transformation of maize callus, immatureembryos are cultured for approximately 8-21 days after excision to allowcallus to develop. Callus is then incubated for about 30 minutes at roomtemperature with the Agrobacterium suspension, followed by removal ofthe liquid by aspiration. The callus and Agrobacterium are co-culturedwithout selection for 3-6 days followed by selection on paromomycin forapproximately 6 weeks, with biweekly transfers to fresh media, andparomomycin resistant callus identified as containing the recombinantDNA in an expression cassette.

For transformation by microprojectile bombardment, immature maizeembryos are isolated and cultured 3-4 days prior to bombardment. Priorto microprojectile bombardment, a suspension of gold particles isprepared onto which the desired recombinant DNA expression cassettes areprecipitated. DNA is introduced into maize cells as described in U.S.Pat. Nos. 5,550,318 and 6,399,861 using the electric discharge particleacceleration gene delivery device. Following microprojectilebombardment, tissue is cultured in the dark at 27 degrees C.

To regenerate transgenic corn plants trangenic callus resulting fromtransformation is placed on media to initiate shoot development inplantlets which are transferred to potting soil for initial growth in agrowth chamber at 26 degrees C. followed by a mist bench beforetransplanting to 5 inch pots where plants are grown to maturity. Theplants are self fertilized and seed is harvested for screening as seed,seedlings or progeny R2 plants or hybrids, e.g., for yield trials in thescreens indicated above.

Example 3

This example further illustrates the production and identification oftransgenic seed for transgenic corn having an enhanced agronomic trait,i.e. enhanced nitrogen use efficiency, increased yield, enhanced wateruse efficiency, enhanced tolerance to cold and/or improved seedcompositions as compared to control plants. Transgenic corn seed andplants comprising recombinant DNA from each of the genes cloned in oneof base vectors as identified in Table 1 are prepared by transformation.Many transgenic events which survive to fertile transgenic plants thatproduce seeds and progeny plants will not exhibit an enhanced agronomictrait. The transgenic plants and seeds having enhanced agronomic traitsof this invention are identified by screening for nitrogen useefficiency, yield, water use efficiency, and cold tolerance. Transgenicplants providing seeds with improved seed compositions are identified byanalyzing for seed compositions including protein, oil and starchlevels.

A. Enhanced Nitrogen Use Efficiency

The transgenic plants with enhanced nitrogen use efficiency provided bythis invention were selected through the selection process according tothe standard procedure described above and the performance of thesetransgenic plants are shown in Table 8 below.

TABLE 8 Leaf chlorophyll area Leaf chlorophyll Shoot fresh mass PercentMean of Percent Mean of Percent Mean of Event ID change Mean controlsP-value change Mean controls P-value change Mean controls P-valueZM_M24857 −1 5366.5 5430 0.75 2 27.8 27.3 0.48 −3 51.6 53.4 0.31ZM_M24857 −24 4150.6 5430 0.00 −8 25.1 27.3 0.01 −33 36 53.4 0.00ZM_M24861 12 3811.5 3397.7 0.00 7 25.2 23.5 0.02 8 31.2 28.8 0.02ZM_M24861 0 5430.4 5430 1.00 6 28.9 27.3 0.04 1 54.2 53.4 0.66 ZM_M24870−2 5347.4 5430 0.68 −1 27 27.3 0.72 −9 48.9 53.4 0.01 ZM_M24870 −35268.1 5430 0.41 5 28.6 27.3 0.10 −5 50.8 53.4 0.14 ZM_M24873 −7 5023.85430 0.04 −9 24.8 27.3 0.00 −18 43.7 53.4 0.00 ZM_M24873 −5 5159.9 54300.17 4 28.4 27.3 0.15 −11 47.7 53.4 0.00 ZM_M24874 −3 5289.5 5430 0.48 227.8 27.3 0.50 −3 51.9 53.4 0.40 ZM_M24874 −2 5319.7 5430 0.58 1 27.527.3 0.77 −2 52.4 53.4 0.58 ZM_M26391 −9 4914.4 5430 0.01 0 27.2 27.30.91 −2 52.5 53.4 0.60 ZM_M26391 −3 5273.7 5430 0.43 3 28 27.3 0.35 −252.2 53.4 0.48

Yield

The transgenic plants with enhanced yield provided by this inventionwere selected through the selection process according to the standardprocedure described above and the performance of these transgenic plantsare shown in Tables 9 and 10 below indicating the change in corn yieldmeasured in bushels per acre.

TABLE 9 Broad Acre Yield High density Event Year 1 Year 2 Yield 248613.9 −2.22 −5.3 24862 0.51 −1.86 2.8 24870 2.33 5.41 7.81 24874 5.21 2.618.21 26391 1.13 −3.59 5.1

TABLE 10 Percent Event Delta change P-value ZM_M81660 −6.20 −3.47 0.05ZM_M81671 −21.99 −12.32 0.00 ZM_M81675 −23.94 −13.41 0.00 ZM_M81677−3.71 −2.08 0.23 ZM_M81682 −5.58 −3.12 0.11 ZM_M81684 −14.72 −8.25 0.00ZM_M81687 4.83 2.71 0.13 ZM_M81688 −14.64 −8.20 0.00

Water Use Efficiency

The transgenic plants with enhanced water use efficiency provided bythis invention were selected through the selection process according tothe standard procedure described above and the performance of thesetransgenic plants are shown in Table 11 below.

TABLE 11 % Pvalue % Pvalue % Pvalue % Pvalue Event SAH SAH RGR RGR SDMSDM RWC RWC ZM_M24857 1.02 0.02 1.63 0.05 3.29 0.02 1.52 0.16 ZM_M24857−4.22 0.00 10.66 0.00 −4.33 0.00 4.59 0.00 ZM_M24861 −1.53 0.00 2.090.01 2.88 0.03 2.65 0.02 ZM_M24861 −2.75 0.00 5.85 0.00 0.33 0.81 4.860.00 ZM_M24862 −0.56 0.20 −5.05 0.00 3.33 0.01 −3.04 0.01 ZM_M24870−3.17 0.00 8.47 0.00 −4.36 0.00 −1.29 0.23 ZM_M24870 0.29 0.50 1.24 0.12−0.36 0.79 −2.05 0.06 ZM_M24873 −3.54 0.00 6.88 0.00 −4.88 0.00 1.300.25 ZM_M24873 −4.61 0.00 10.51 0.00 −3.08 0.02 −1.92 0.08 ZM_M248740.00 1.00 −3.57 0.00 2.96 0.03 −2.45 0.03 ZM_M24874 −1.96 0.00 2.17 0.01−0.60 0.66 1.16 0.31 ZM_M26391 −2.18 0.00 4.02 0.00 −1.01 0.45 −0.110.92 ZM_M26391 0.76 0.08 −4.44 0.00 2.77 0.04 2.67 0.01

Cold Tolerance

The transgenic plants with enhanced cold tolerance provided by thisinvention were selected through the selection process according to thestandard procedure described above and the performance of the earlyseedling growth of these transgenic plants are shown in Table 12 below.

TABLE 12 Root length Shoot length Seedlling length Percent Mean ofPercent Mean of Percent Mean of Event ID change Mean controls P-valuechange Mean controls P-value change Mean controls P-value ZM_M24857 2314.81 12.07 0.01 15 10.07 8.77 0.02 19 24.89 20.84 0.01 ZM_M24857 1814.1 11.97 0.01 6 10.35 9.72 0.13 13 24.45 21.69 0.02 ZM_M24857 9 13.6912.56 0.03 12 9.13 8.17 0.01 10 22.81 20.74 0.01 ZM_M24857 14 13.6811.97 0.04 10 10.66 9.72 0.02 12 24.33 21.69 0.03 ZM_M24857 −11 10.1211.39 0.10 −3 8.24 8.48 0.64 −8 18.36 19.87 0.21 ZM_M24861 5 13.43 12.790.32 −10 7.71 8.58 0.07 −1 21.13 21.37 0.82 ZM_M24861 4 12.4 11.97 0.61−3 9.43 9.72 0.48 1 21.83 21.69 0.91 ZM_M24861 −10 10.15 11.32 0.11 −128.96 10.22 0.01 −11 19.11 21.54 0.04 ZM_M24862 −9 10.32 11.32 0.17 −79.47 10.22 0.14 −8 19.79 21.54 0.13 ZM_M24870 14 13.65 11.97 0.05 710.43 9.72 0.09 11 24.09 21.69 0.04 ZM_M24870 −2 12.28 12.56 0.59 1 8.298.17 0.75 −1 20.58 20.74 0.83 ZM_M24870 11 13.31 11.97 0.11 4 10.11 9.720.34 8 23.42 21.69 0.14 ZM_M24870 0 10.46 10.45 0.98 2 8.08 7.96 0.82 118.55 18.41 0.89 ZM_M24873 10 13.2 11.97 0.14 5 10.2 9.72 0.25 8 23.3921.69 0.15 ZM_M24873 −8 11.83 12.79 0.13 −10 7.75 8.58 0.08 −8 19.5821.37 0.08 ZM_M24873 17 14.06 11.97 0.01 16 11.3 9.72 0.00 17 25.3621.69 0.00 ZM_M24873 −7 11.74 12.56 0.11 0 8.16 8.17 0.98 −4 19.91 20.740.28 ZM_M24874 −13 11.15 12.79 0.01 −19 6.92 8.58 0.00 −15 18.07 21.370.00 ZM_M24874 13 13.52 11.97 0.07 8 10.54 9.72 0.05 11 24.06 21.69 0.05ZM_M24874 −10 11.33 12.56 0.02 −4 7.87 8.17 0.43 −7 19.21 20.74 0.05ZM_M24874 2 12.25 11.97 0.74 7 10.39 9.72 0.11 4 22.64 21.69 0.42ZM_M26391 23 14.72 11.97 0.00 17 11.37 9.72 0.00 20 26.08 21.69 0.00ZM_M26391 −6 11.82 12.56 0.15 7 8.72 8.17 0.16 −1 20.54 20.74 0.80ZM_M26391 −23 8.09 10.45 0.00 −14 6.88 7.96 0.04 −19 14.97 18.41 0.00ZM_M26391 9 13.01 11.97 0.21 10 10.72 9.72 0.02 9 23.72 21.69 0.09

TABLE 13 Oil Y2 Hybrid Data Control Percent Y1 Hybrid Data EventConstruct Mean mean change Delta P-value Delta P-value ZM_M24870PMON68392 4.48 4.29 4.28 0.18 0.04 0.14 0.15 ZM_S68719 PMON74775 4.434.12 7.38 0.30 0.00 #N/A #N/A ZM_S69656 PMON74775 4.36 4.12 5.59 0.230.03 0.33 0.02

Improved Seed Composition

The transgenic plants with improved seed composition provided by thisinvention were selected through the selection process according to thestandard procedure described above and the performance of thesetransgenic plants are shown in Tables 13-5.

TABLE 14 Oil Mean Event Construct Mean control Delta P-value ZM_M92534PMON84131 4.94 4.51 0.42 0.00 ZM_M91731 PMON84131 4.90 4.51 0.38 0.01ZM_M92532 PMON84131 4.87 4.51 0.35 0.02

TABLE 15 Protein Protein Protein Event Construct delta p-value ZM_M24870PMON68392 0.44 0.02 ZM_S68719 PMON74775 0.35 0.12 ZM_S69656 PMON747750.21 0.35

Example 4

This example illustrate transgenic plants with enhanced traits throughcombinations. As illustrated in the Example 3, transgenic plants withenhanced agronomic traits are generated employing the recombinant DNAfrom each of the genes identified in Table 1. To produce furtherenhancement of agronomic traits in transgenic plants, the genes of Table1 are combined with one or more additional genes that enhance agronomictraits to generate a transgenic plant with greater enhancement in one ormore agronomic traits than either gene alone. This combination isachieved through either through transformation or breeding. Thefollowing example illustrates this principle. A transgenic maize plantstably transformed with a construct, pMON74923, containing the Zea maysphytochrome B (phyB) gene (SEQ ID NO: 15) under the control of a maizealdolase (FDA) promoter (U.S. patent application Ser. No. 09/757,089)was crossed with a transgenic maize plant stably transformed withpMON68392. The cross demonstrated an increased yield (bu./a) of 7.2%compared to the maize plant containing the phyB gene alone (2.4%).

Example 5 Soybean Plant Transformation

This example illustrates plant transformation useful in producing thetransgenic soybean plants of this invention and the production andidentification of transgenic seed for transgenic soybean having anenhanced agronomic trait, i.e. enhanced nitrogen use efficiency,enhanced yield, enhanced water use efficiency, enhanced growth undercold stress, and/or enhanced seed oil, protein and/or starch levels ascompared to control plants. For Agrobacterium mediated transformation,soybean seeds are germinated overnight and the meristem explantsexcised. The meristems and the explants are placed in a wounding vessel.Soybean explants and induced Agrobacterium cells from a straincontaining plasmid DNA with the gene of interest cassette and a plantselectable marker cassette are mixed no later than 14 hours from thetime of initiation of seed germination and wounded using sonication.Following wounding, explants are placed in co-culture for 2-5 days atwhich point they are transferred to selection media for 6-8 weeks toallow selection and growth of transgenic shoots. Trait positive shootsare harvested approximately 6-8 weeks post bombardment and placed intoselective rooting media for 2-3 weeks. Shoots producing roots aretransferred to the greenhouse and potted in soil. Shoots that remainhealthy on selection, but do not produce roots are transferred tonon-selective rooting media for an additional two weeks. Roots from anyshoots that produce roots off selection are tested for expression of theplant selectable marker before they are transferred to the greenhouseand potted in soil.

Example 6

This example further illustrates the production and identification oftransgenic seed for transgenic soybean having an enhanced agronomictrait, i.e. enhanced nitrogen use efficiency, increased yield, enhancedwater use efficiency, enhanced growth under cold stress, and/or improvedseed compositions as compared to control plants. Transgenic soybean seedand plants comprising recombinant DNA from each of the genes cloned inone of base vectors as identified in Table 1 are prepared bytransformation. Many transgenic events which survive to fertiletransgenic plants that produce seeds and progeny plants will not exhibitan enhanced agronomic trait. The transgenic plants and seeds havingenhanced agronomic traits of this invention are identified by screeningfor nitrogen use efficiency, yield, water use efficiency, and coldtolerance. Transgenic plants providing seeds with improved seedcompositions are identified by analyzing for seed compositions includingprotein, oil and starch levels.

What is claimed is:
 1. A plant cell with stably integrated, recombinant DNA comprising a promoter that is functional in plant cells and that is operably linked to DNA that encodes a protein having domains of amino acids in a sequence that exceed the Pfam gathering cutoff for amino acid sequence alignment with a Pfam Homeobox protein domain family and a Pfam HALZ protein domain family; wherein the Pfam gathering cuttoff for the Homeobox protein domain family is −4 and the and the Pfam gathering cuttoff for the HALZ protein domain family is 17; wherein said plant cell is selected from a population of plant cells with said recombinant DNA by screening plants that are regenerated from plant cells in said population and that express said protein for an enhanced trait as compared to control plants that do not have said recombinant DNA; and wherein said enhanced trait is selected from group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.
 2. A plant cell of claim 1 wherein said protein has an amino acid sequence with at least 90% identity to a consensus amino acid sequence in the group of consensus amino acid sequences consisting of the consensus amino acid sequence constructed from an alignment of SEQ ID NO:5 through SEQ ID NO:8.
 3. A plant cell of claim 1 wherein said protein is selected from the group of proteins having an amino acid sequence of SEQ ID NO:5 through SEQ ID NO:8.
 4. A plant cell of claim 1 further comprising DNA expressing a protein that provides tolerance from exposure to an herbicide applied at levels that are lethal to a wild type of said plant cell.
 5. A plant cell of claim 4 wherein the agent of said herbicide is a glyphosate, dicamba, or glufosinate compound.
 6. A transgenic plant comprising a plurality of the plant cell of claim
 1. 7. A transgenic plant of claim 6 which is homozygous for said recombinant DNA.
 8. A transgenic seed comprising a plurality of the plant cell of claim
 1. 9. A transgenic seed of claim 8 from a corn, soybean, cotton, canola, alfalfa, wheat or rice plant.
 10. Non-natural, transgenic corn seed of claim 9 wherein said seed can produce corn plants that are resistant to disease from the Mal de Rio Cuarto virus or the Puccina sorghi fungus or both.
 11. A transgenic pollen grain comprising a haploid derivative of a plant cell of claim
 1. 12. A method for manufacturing non-natural, transgenic seed that can be used to produce a crop of transgenic plants with an enhanced trait resulting from expression of stably-integrated, recombinant DNA comprising a promoter that is (a) functional in plant cells and (b) is operably linked to operably linked to DNA that encodes a protein having domains of amino acids in a sequence that exceed the Pfam gathering cutoff for amino acid sequence alignment with a Pfam Homeobox protein domain family and a Pfam HALZ protein domain family; wherein the Pfam gathering cuttoff for the Homeobox protein domain family is −4 and the and the Pfam gathering cuttoff for the HALZ protein domain family is 17; and wherein said enhanced trait is selected from the group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil, said method for manufacturing said seed comprising: (a) screening a population of plants for said enhanced trait and said recombinant DNA, wherein individual plants in said population can exhibit said trait at a level less than, essentially the same as or greater than the level that said trait is exhibited in control plants which do not express the recombinant DNA, (b) selecting from said population one or more plants that exhibit the trait at a level greater than the level that said trait is exhibited in control plants, (c) verifying that said recombinant DNA is stably integrated in said selected plants, (d) analyzing tissue of a selected plant to determine the production of a protein having the function of a protein encoded by nucleotides in a sequence of one of SEQ ID NO:1-4; and (e) collecting seed from a selected plant.
 13. A method of claim 12 wherein plants in said population further comprise DNA expressing a protein that provides tolerance to exposure to an herbicide applied at levels that are lethal to wild type plant cells, and wherein said selecting is effected by treating said population with said herbicide.
 14. A method of claim 13 wherein said herbicide comprises a glyphosate, dicamba, or glufosinate compound.
 15. A method of claim 12 wherein said selecting is effected by identifying plants with said enhanced trait.
 16. A method of claim 12 wherein said seed is corn, soybean, cotton, alfalfa, wheat or rice seed.
 17. A method of producing hybrid corn seed comprising: (a) acquiring hybrid corn seed from a herbicide tolerant corn plant which also has stably-integrated, recombinant DNA comprising a promoter that is (a) functional in plant cells and (b) is operably linked to operably linked to DNA that encodes a protein having domains of amino acids in a sequence that exceed the Pfam gathering cutoff for amino acid sequence alignment with a Pfam Homeobox protein domain family and a Pfam HALZ protein domain family; wherein the Pfam gathering cuttoff for the Homeobox protein domain family is −4 and the and the Pfam gathering cuttoff for the HALZ protein domain family is 17; (b) producing corn plants from said hybrid corn seed, wherein a fraction of the plants produced from said hybrid corn seed is homozygous for said recombinant DNA, a fraction of the plants produced from said hybrid corn seed is hemizygous for said recombinant DNA, and a fraction of the plants produced from said hybrid corn seed has none of said recombinant DNA; (c) selecting corn plants which are homozygous and hemizygous for said recombinant DNA by treating with an herbicide; (d) collecting seed from herbicide-treated-surviving corn plants and planting said seed to produce further progeny corn plants; (e) repeating steps (c) and (d) at least once to produce an inbred corn line; (f) crossing said inbred corn line with a second corn line to produce hybrid seed.
 18. The method of selecting a plant comprising cells of claim 1 wherein an immunoreactive antibody is used to detect the presence of said protein in seed or plant tissue.
 19. Anti-counterfeit milled seed having, as an indication of origin, a plant cell of claim
 1. 20. A method of growing a corn, cotton or soybean crop without irrigation water comprising planting seed having plant cells of claim 1 which are selected for enhanced water use efficiency.
 21. A method of claim 20 comprising providing up to 300 millimeters of ground water during the production of said crop.
 22. A method of growing a corn, cotton or soybean crop without added nitrogen fertilizer comprising planting seed having plant cells of claim 1 which are selected for enhanced nitrogen use efficiency. 